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| United States Patent |
5,273,715
|
|
Bridgham
, et al.
|
December 28, 1993
|
Automated system for providing a sequence of chemicals to a reaction
process
Abstract
An automated system for providing a preselected sequence of chemicals to a
reaction. This apparatus includes a track on which a set of cartridges are
placed in a preselected order corresponding to an order in which they are
to be used in a reaction process. These cartridges are moved past a point
at which these chemicals are extracted for use in the process. The
chemicals are preferably in liquid form and are contained in containing
having a top seal through which a needle can penetrate to extract
chemicals for use in the process. These containers preferably contain the
aliquot portion needed for the process, thereby providing a mechanism for
providing accurate amounts of each chemical to the process.
| Inventors:
|
Bridgham; John (Palo Alto, CA);
Geiser; Timothy G. (La Honda, CA);
Hunkapiller; Michael W. (San Carlos, CA);
Kent; Stephen B. H. (Pasadena, CA);
Marplott; Mark P. (Los Altos, CA);
Ramstad; Paul O. (Oakland, CA)
|
| Assignee:
|
Applied Biosystems, Inc. (Foster City, CA)
|
| Appl. No.:
|
973137 |
| Filed:
|
November 6, 1992 |
| Current U.S. Class: |
422/63; 422/65; 422/67; 422/100; 422/101 |
| Intern'l Class: |
G01N 021/00; G01N 031/00; B01L 003/02; B01L 011/00 |
| Field of Search: |
422/63,67,65,100,101
|
References Cited
U.S. Patent Documents
| 3531258 | Sep., 1970 | Merrifield et al. | 23/252.
|
| 3557077 | Jan., 1971 | Brunfeldt | 260/112.
|
| 3647390 | Mar., 1972 | Kubodera et al. | 422/116.
|
| 4668476 | May., 1987 | Bridgham et al. | 422/62.
|
| 4816513 | Mar., 1989 | Bridgham et al. | 525/54.
|
Primary Examiner: Housel; James C.
Assistant Examiner: Bhat; N.
Attorney, Agent or Firm: Smith; Joseph H., Frazzini; John A.
Parent Case Text
This case is a divisional of U.S. application Ser. No. 07/328,488, filed on
Mar. 24, 1989 by John D. Bridgham, et al. entitled Automated Polypeptide
Synthesis Apparatus, now U.S. Pat. No. 5,186,898 which was a divisional of
U.S. application Ser. No. 53,324 filed May 22, 1987 by John D. Bridgham,
et al entitled Automated Polypeptide Synthesis Apparatus and issued as
U.S. Pat. No. 4,816,513 on Mar. 28, 1989; which was a divisional of U.S.
application Ser. No. 592,638 filed Mar. 23, 1984 by John D. Bridgham, et
al entitled Automated Polypeptide Synthesis Apparatus, issued as U.S. Pat.
No. 4,668,476 on May 26, 1987.
Claims
What is claimed is:
1. An automated system for providing a preselected sequence of chemicals to
a reaction, said apparatus comprising:
a track for holding a linear array of cartridges and for directing the
motion of said cartridges along said track, at least two of said
cartridges each containing an associated reagent;
means for extracting reagent from a cartridge at a preselected position
along said track at which such extraction is to occur; and
means for controllably moving said array of cartridges along said track
such that each of these cartridges can be moved to said preselected
position at which extraction can occur.
2. An automated system as in claim 1 wherein each cartridge includes a bar
code, said system further comprising:
bar code reader positioned to read said bar code on each cartridge as that
cartridge moves past said bar code reader.
3. An automated system as in claim 2 further comprising:
a computer that is responsive to a user input sequence of chemicals to be
utilized and to information from said bar code reader to ensure that a
sequence of reagents extracted from said cartridges complies with said
user input sequence.
4. An automated system as in claim 1 wherein each cartridge contains a
preselected amount of reagent and wherein said means for extracting
reagent extracts substantially all of the reagent in each cartridge from
which it extracts any reagent, whereby an accurate amount of reagent is
provided in each successive step of extracting reagent from a cartridge.
5. An automated system as in claim 4 wherein said means for extracting
includes a first needle;
each of said cartridges has a top portion sealed by a membrane that is
penetrated by said first needle when a reagent is extracted from that
cartridge and wherein a bottom portion of said cartridge narrows down to
an apex into which said first needle is inserted during extraction of
liquid from said cartridge, whereby substantially all of the liquid on
said cartridge can be extracted, thereby enabling a dose of said reagent
utilized in reaction to be accurately by the dose supplied in the
cartridge containing that dose.
6. An automated system as in claim 5 wherein said track includes and end
wall against which a cartridge is pressed when liquid is to be extracted
from that cartridge, said system further comprising:
means for pressing on a last cartridge in said linear array to press a
first cartridge in said linear array against said end wall such that said
first cartridge is aligned with said first needles such that said first
needle is aligned over said apex of the first cartridge.
7. An automated system as in claim 6 further comprising:
means for ejecting from said array the cartridge that is pressed against
said end wall, whereby a subsequent cartridge in the array is then pressed
against said end wall.
8. An automated system as in claim 5 further comprising:
a second needle through which a gas can be forced into a cartridge to push
liquid within this cartridge out of the cartridge through said first
needle.
Description
FIELD OF INVENTION
This invention relates to apparatus for the automated synthesis of
polypeptides, and particularly to apparatus for automatically pre-forming
activated species of (alpha-amino protected) amino acids immediately prior
to introduction into solid phase synthesis reactions.
BACKGROUND OF THE INVENTION
Since its inception in 1962, R. B. Merrifield's concept of solid phase
peptide synthesis has seen many improvements and has now become an
established technique in the art. Literally hundreds of investigations
have been published describing the chemical details of the method (See for
example, Merrifield, R. B.: Science 150, 178 (1965); Merrifield, R. B.:
Sci. Amer. 218, 56 (1968); Stewart, J. M., Young, J. D.: In: Solid Phase
Peptide Synthesis. San Francisco, Calif.: Freeman 1969; and Erickson, B.
W., Merrifield, R. B.: In: The Proteins (eds. Neurath, R. L. Hill), III.
Ed., Vol. 2, pp 255-527. New York: Academic Press 1976).)
Typically, solid phase peptide synthesis begins with the covalent
attachment of the carboxyl end of an (alpha-amino protected) first amino
acid in the peptide sequence through an organic linker to an insoluble
resin bead (typically 25-300 microns in diameter), illustrated by:
##STR1##
A general cycle of synthesis then consists of deprotection of the resin
bound alpha-amino group, washing (and neutralization if necessary),
followed by reaction with with some carboxyl activated form of the next
(alpha-amino protected) amino acid to yield:
##STR2##
Repetition of the cycle to the n.sup.th amino acid then yields:
##STR3##
At the end of the synthesis, the link of the peptide to its polymer
support is cleaved, and the dissolved peptide is separated from the
insoluble resin and purified.
Although this process is simple in principle, in practice it can be quite
difficult to obtain peptides over about 30 amino acids long which have any
substantial purity. The reason for this is that the average step yield has
a profound effect on the purity of the product peptide, as illustrated by
the values in the following table for synthesis of a 30 amino acid
peptide.
TABLE
______________________________________
30 AMINO ACID PEPTIDE
Average Step Yield (%)
Product Purity (%)
______________________________________
95.0 21
99.5 86
99.7 91
______________________________________
The results are even more problematic for longer peptides, eg. for a target
peptide with 101 residues, a step yield of 99.0% provides a product of
only 36% purity. In all cases, the by-products of peptide synthesis
consist of a complex mixture of molecules which are chemically similar to
the target peptide. Chromatographic purification can be extraordinarily
difficult and time consuming as the relative amount of by-product
molecules begins to exceed about 25%.
The efficiency of step yield is dependent on many factors such as the
nature and quality of the protected amino acids, solvent purity, chemical
integrity of the resin, the chemical nature of the organic linker, the
form of the activated carboxyl of the amino acid, efficiency of the wash
steps, the synthesis protocol, and in some instances the identity of an
amino acid in conjunction with a particular sequence segment to which it
is being added.
Each of the above factors, when not optimally controlled, will contribute
some significant increment to yield reduction in every coupling step. At
the present time, the complexity of these factors is such that average
step yields in solid phase peptide synthesis are typically in the range of
93-97% for both manual and automated executions. For practical
applications on a commercially reasonable scale, such as for the
development of pharmaceuticals, enzyme substrates and inhibitors,
hormones, vaccines, and diagnostic reagents, such low step yields
significantly increase costs of production and in many cases make such
direct solid phase synthesis of peptides impractical.
Prior art peptide synthesizers operate essentially as "washing machines"
which automate the monotonous fluid manipulations of deprotection,
addition of coupling agent, and washing. In no case do existing commercial
peptide synthesizers form an activated amino acid species outside or
independent of the reaction vessel. Typically, protected amino acid and
DCC are added to the reaction vessel containing the resin bound, incipient
peptide chain so that activation of the amino acid occurs in the presence
of the deprotected alpha-amino group. This approach both limits the
possibility (or feasibility) of optimizing activation conditions for
individual amino acids and requires that any modification of activation
conditions be done in the presence of the deprotected alpha-amino group
and the growing, resin-bound peptide chain. This fact makes it difficult,
if not impossible, to optimize activation parameters by analyzing rates of
formation and relative thermal and solvent stabilities of the individual,
activated amino acid species. Additionally, the ability to use various
thermal inputs during the activation process can only be done in the
presence of the peptide chain.
SUMMARY OF THE INVENTION
In accordance with the preferred embodiment of the invention, an apparatus
is provided for automatically constructing a polypeptide of high purity,
up to 50 amino acids in length, using only single couplings. The apparatus
includes an activation system for receiving protected amino acids, one
kind at a time, having a common vessel (an activator vessel) in which to
activate each of the amino acids in the order received to form a sequence
of aliquots of activated species of each of the amino acids, each aliquot
containing one kind of amino acid and the sequence of aliquots of each
kind of amino acid being in the order desired in the peptide. Also
included is a reaction vessel for containing a resin used in solid-phase
peptide synthesis for attaching a peptide chain thereto. A transfer system
is also provided, which operates under control of a computer, to transfer
the activated species from the activation system to the reaction vessel
and to transfer amino acids, reagents, gases, and solvents from one part
of the apparatus to another. The activator system also includes a
temperature controlled concentrator vessel in which an activator solvent,
which is used in the activator vessel when creating the activated species
of the amino acid, is replaced by a coupling solvent to enhance the
coupling of the activated species to the peptide chain in the reaction
vessel. This replacement is accomplished a short period of time (typically
less than thirty minutes) before the activated amino acid is introduced
into the reaction vessel, by adding the coupling solvent to the
concentrator vessel together with the activated species and the activator
solvent, and sparging gas through the resulting solution to selectively
evaporate the activator solvent, activator solvent being chosen with a
boiling point lower than the boiling point of the coupling solvent. The
concentrator is heated as necessary to replace heat lost by evaporation.
Also included in the synthesizer system is a vortexer for affecting total
washing of materials in the reaction vessel and the reaction vessel
itself, an automated peptide resin sampling system, and an autodelivery
system for providing individual containers of amino acid to the
synthesizer in the order desired in the peptide sequence. Also, a
specialized container for use in the autodelivery system is provided which
has a vee-shaped bottom in order to permit extraction of as much amino
acid as is practicable which permits precise control over stoichiometry. A
liquid sensor system is also included to monitor transitions between gases
and liquids in specific tubes in the synthesizer in order to provide input
signals to the computer system for control purposes.
The computer system software which controls the operation of the
synthesizer is organized according to a series of menus which allows the
user of the system to select individual cycles of operation for each
vessel in the synthesizer. In addition each cycle can be user-defined into
a series of functions, each of which corresponds to a standard set of
instructions for individual valves and other switched systems associated
with the synthesizer. Also, using the menu system, the user can define
alternative individual functions as well.
In addition to the menu driven control system, an algorithm has been
developed which is related to the organization of the computer software
into individual cycles for each vessel. Operating the synthesizer
according to the algorithm provides for optimum efficiency in the
production of a peptide for any given selection of cycles.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1a and 1b illustrate the fluid delivery system of the apparatus
according to the invention.
FIG. 1A illustrates how FIGS. 1a and 1b are to be arranged to illustrate
the fluid delivery system.
FIG. 1B illustrates gas sources that are connected through manifolds and
regulators to indicated inputs of the elements of FIGS. 1a and 1b.
FIG. 2 illustrates the computer system used for controlling the apparatus.
FIG. 3 is a top view of an autodelivery system for providing individual
containers to the synthesis apparatus.
FIG. 4 is a cross-sectional view of a reaction vessel according to the
invention showing the results of vortexing on fluids contained therein.
FIGS. 5a, 5b, and 5c are three views of a container used in the
autodelivery system.
FIG. 5d is a bottom view of the container illustrated in FIGS. 5a-5c.
FIG. 6 is a table showing the dimensions of the container used in the
autodelivery system.
FIGS. 7a and 7b show two views of a liquid sensor used in the apparatus.
FIGS. 8-16 illustrate various menus used in the computer system to define
operations according to which to run the synthesizer apparatus.
FIG. 17a and 17b illustrate the menu flow scheme.
FIG. 17 illustrates how FIGS. 17a and 17b are to be arranged to illustrate
the menu flow scheme.
FIG. 18 is a diagram to provide definitions for use in the calculational
scheme for optimizing production of peptide.
FIG. 19 provides an example of a calculation for optimizing the production
of a peptide which requires three cycles of coupling.
FIGS. 20as and 20b compare prior yields with the yields produced by the
apparatus disclosed herein.
DETAILED DESCRIPTION OF THE INVENTION
Before the details of the apparatus of the invention are described, it is
useful first to understand the chemical approach of the solid phase
peptide synthesis which the apparatus is designed to optimize.
For the present invention, the preferred mode of the synthesis takes place
primarily in three phases. The first, or activation phase involves the
production of the (alpha-amino protected) amino acid symmetric anhydride
as the acylating species:
##STR4##
Symmetric anhydrides are extremely effective, activated carboxyl forms of
amino acids, since their couplings are substantially free of racemization
in the absence of base and since quantitative single couplings are usually
assured for most amino acid additions, with the exception of asparagine,
glutamine, and arginine--each of which is more efficiently coupled by an
alternative activation method which will be discussed later. The generally
quantitative nature of couplings with symmetric anhydrides makes them most
useful where automated synthesis precludes convenient step by step
quantitative monitoring. The apparatus herein described automatically
synthesizes symmetric anhydrides immediately before incorporation into the
peptide chain. Because of the marginal stability of symmetric anhydrides
and their difficulty of isolation in pure form, their use in the past has
been limited to manual preformation followed by introduction into an
automated synthesis machine.
The procedure for synthesizing pre-formed symmetric anhydrides (PSA's)
consists of reacting 0.5 equivalents of dicyclohexylcarbodiimide (DCC)
with 1.0 equivalent of protected amino acid in dichloromethane (DCM)
according to the equation:
##STR5##
dichloromethane being an optimal solvent for the synthesis of PSA's,
particularly where the alpha-amino protecting group P is the
t-butyloxycarbonyl group (t-BOC). The DCU, formed in the reaction,
however, is very insoluble in dichloromethane and precipitates during the
PSA reaction. After completion of the reaction, the PSA/DCM solution is
filtered away from the DCU precipitate, and the second, or concentration
phase is begun. In the concentration phase the DCM is removed and replaced
by polar aprotic solvent, preferably N,N-dimethylformamide (DMF), to
enhance coupling efficiency during later solid phase reactions. The third,
or reaction phase then follows the general schema described in the
Background of the Invention whereby to, attach an additional amino acid to
the sequence the carboxyl end of the PSA is reacted with an alpha-amino
deprotected resin-bound peptide chain.
Apparatus
In accordance with the preferred embodiment of the invention, an apparatus
for achieving the synthesis described above is illustrated in FIGS. 1a,
1b, 2 and 3, which show respectively a fluid delivery system, for routing
the various amino acids, reagents, solvents and gases throughout the
apparatus; a computer system for effecting automatic control over the
numerous switches which control the valves, sensors, temperature of
certain vessels, and motors in the apparatus; and an autodelivery system
for transporting protected amino acids to the apparatus in the order
desired in the peptide sequence.
The fluid delivery system includes three primary vessels: an activator
vessel 11, where the PSA is formed; a concentrator vessel 13, where the
PSA/DCM solution from the activator vessel is transferred so that DCM may
be replaced by DMF to enhance coupling; and a reaction vessel 15 which
contains the growing resin-bound peptide chain.
Activator vessel 11 is typically cylindrical, about 40 ml in volume, and is
preferably constructed of glass in order for the operator of the device to
visually inspect the progress of reactions or cleaning cycles. At the
bottom of the activator vessel is a glass frit 17 of coarse poresize which
is used to filter the DCU precipitate from the PSA/DCM solution when
transferring the solution to the concentrator vessel 13. Activator vessel
11 also contains an overhead nozzle 19 which faces upward in order to
achieve a total washdown of the headspace and walls after each amino acid
is transferred out of the vessel. Activator 11 is coupled to the
autodelivery system and to various gases and reagents as shown via a valve
block 23, which is an assembly of zero dead volume valves such as that
described in U.S. Pat. No. 4,008,736, issued Feb. 22, 1977, entitled VALVE
ARRANGEMENT FOR DISTRIBUTING FLUIDS, by Wittman-Liebold, et al., as are
all other valve blocks in the system. Valve block 23 is operated under the
control of the computer system, as are all other valve blocks and gas
valves in the apparatus. Activator 11 is coupled via nozzle 19 to another
valve block 25 which controls the flow of methanol and DCM into the vessel
for dissolving DCU precipitate for cleaning and which controls the
pressure inside the vessel to effect transfers of materials into and out
of the vessel from block valve 23. Transfers from the bottom of the vessel
take place through a translucent tube 29, typically constructed of
Teflon.TM., the transfers being monitored by the computer system by means
of a liquid sensor 27 which detects transitions in tube 29 between gases
and liquids. Typically tube 29, and other tubes in the system to which
similar liquid sensors are attached have a roughly calibrated flow
resistance and operate at a fixed known pressure during transfers, so that
the length of time required for a transfer corresponds directly to the
volume of material which is transferred. For DCC and HOBT, which require
specific volumes, a calibrated delivery loop is used to achieve a higher
accuracy. Hence, the computer system can accurately monitor all flow into
and out of the activator vessel. Although details of the liquid sensor
will be described later, it is important to emphasize that the use of
liquid sensors is not required for operation of the apparatus. They are
useful however in achieving better system control.
The next major section of the fluid delivery system is the concentrator
vessel 13. Its construction is substantially the same as that of activator
vessel 11, and includes an overhead nozzle 31, and a glass filter frit 35
at the bottom. In addition, however, the concentrator vessel also has a
band heater 37 attached thereto, which is used to control the temperatures
inside the vessel through the use of a thermistor as the DCM in the
PSA/DCM solution is replaced by DMF. Attached to concentrator vessel 13 is
a valve block 33, and a translucent transfer tube 39 monitored by a liquid
sensor 40. Transfers through tube 39 are controlled by the computer system
by means of valve block 41.
The next major section of the fluid delivery system is the reaction vessel
15, which in the preferred embodiment is a right circular cylinder
oriented vertically and is constructed of a machined fluorocarbon polymer,
such as Teflon.TM., or KEL-F.TM.. The vessel is valved at the top by a
valve block 43 and at the bottom by a valve block 45, each valve being
isolated by a filter such as membrane 47 and membrane 49 which are
typically constructed of a material such as ZITEX.TM., produced by
Chemplast Inc. of Wayne, N.J., although glass frits could also be used.
The reaction vessel is designed to be opened conveniently, both for
initial charging with the loaded resin and for periodic removal of sample
aliquots. This is accomplished by threading the top and bottom of the
reaction vessel cylinder to accommodate threaded caps. The threaded caps
are also used to hold the membranes in place, and each cap is configured
to accept a tube in order to transfer fluids into and out of the reaction
vessel. A typical volume for the reaction vessel can vary widely,
depending on the length of the peptide chain to be synthesized and the
weight and amino acid loading of the synthesis resin. For example, for
chains up to 50 amino acid units in length, starting with 0.5 grams of
resin (0.5 m mole of amino acid), a preferred size is about 40 ml. Those
skilled in the art of solid phase synthesis will realize that the resin
may swell from three to five fold due to solvent imbibement during
synthesis. Mass increase as a result of growth of the peptide chain can
cause an increase in the occupied volume of the reaction vessel from 10%
initially to as much as 80% at the end of the synthesis, depending on the
length of the peptide.
In order to promote efficient coupling and to avoid agglomeration of the
resin beads it is important to agitate the reaction vessel at various
stages in the reaction cycle. Also, it is especially important that the
entire inner surface of reaction vessel 15 be completely rinsed during
each wash cycle between the additions of PSA's from the concentrator
vessel. To achieve this agitation, the bottom of the reaction vessel is
moved in a circle having a radius of about 0.093 in., about its center
axis at about 1500 rpm, by a motor 48 (connected by a pully to an
eccentric on the bottom of the reaction vessel) under control of the
computer system, while the center of the top of the reaction vessel is
held substantially fixed, with the vessel itself being prevented from
rotating. The result is a conically rotational motion of the fluid resin
mixture in the reaction vessel about the vertical axis which has the
appearance of a vortex. This "vortex" agitation mode enables use of very
small volume increments of wash solvent for all washing operations, thus
greatly improving the efficiency of removal of spent reagents and reagent
by-products from the synthesis resin, since it is much more efficient to
extract these materials by successive partitioning, than by a single
extraction. The ability to use small volume increments is a result of the
angular momentum of the fluid resin mixture imparted by the conical
motion. This "vortexing" action creates a distribution of fluid in the
vessel as depicted in FIG. 4, wherein the fluid in the reaction vessel can
be made to contact all interior surfaces of the reaction vessel, for very
small volume increments of the solvent by proper choice of the speed of
rotation. The result is more efficient washing of the resin by smaller
volumes of expensive solvents.
Additionally, this mode of agitation prevents resin agglomeration and
allows total fluid-resin interaction without the use of impeller type
mechanical agitation. With mechanical agitation, the shear and resin
abrasion caused by the impeller can fracture the resin beads into smaller
and smaller particles which can eventually clog the filters, such as
membranes 47 and 49, thus forcing interruption of the synthesis process.
Such an interruption can have dire effects on synthesis, e.g. restriction
of flow (out of the Reaction vessel) could occur during an acid
deprotection step, thereby subjecting the resin bound peptide to the
degradative effects of overlong acid exposure. With the vortex agitator
there are no impeller type shear or abrasive effects on the resin beads.
Those skilled in the art will understand that although in the preferred
embodiment the reaction vessel has been constructed in the shape of a
right circular cylinder, other shapes can also be used, provided they are
not antagonistic to the relatively smooth swirling of the fluid in the
vessel, e.g. a shape such as that of a wine glass appears to have some
desirable properties for the washing cycle. Also, for maximum efficiency,
the apparatus is typically implemented with three reaction vessels 15
while using only one concentrator vessel 13 and one activator vessel 11,
with the fluid distribution from these latter two vessels appropriately
valved to operate with each of the three reaction vessels and their
corresponding valve blocks 43 and 45. It should be understood, however,
that each of these reaction vessels corresponds to a separate sequential
process for creating a peptide, i.e. the first peptide is formed in the
first reaction vessel, then the second peptide is formed in the second
reaction vessel, then the third peptide is formed in the third vessel.
In order to monitor the progress of the synthesis in the reaction vessel, a
resin sampler system 51 is provided. The sampler includes a tube 53
connected into the side of the reaction vessel for extracting materials
therefrom, the flow through the tube being controlled by a computer
controlled valve 55, typically a solenoid operated pinch valve. Tube 53
extends into the bottom portion of resin sampler reservoir 57 which has a
membrane 59, typically ZITEX.TM. located at the top. Also connected to the
top of reservoir 57, on the opposite side of membrane 59, is a tube 61
which is connected to valve block 45. From the bottom of the reservoir
extends another tube 65, which is controlled by a valve 67 also typically
a solenoid operated pinch valve (to achieve zero dead volume), for
collecting fractions from the reservoir.
The gas distribution system for achieving the desired transfers within the
apparatus is illustrated in both FIGS. 1A and 1B, and consists of three
gas manifolds, manifold C, manifold CC, and manifold D, and accompanying
regulators for controlling the distribution from bottles and through the
valve blocks. Also included are two smaller manifolds, manifold 70 for
distributing DCM throughout the apparatus and manifold 71 for distributing
DMF to the activator vessel and the concentrator vessel. Gas flows
throughout the system are controlled by the computer system by means of
solenoid operated valves such as valve 73; (e.g. such as fluorocarbon
valves are made by Angar, Incorporated.) and by the valve blocks already
discussed.
FIG. 2 shows a schematic representation of the computer system which
consists of a microprocessor based microcomputer 81 having an arithmetic
unit 87; a mass storage device 84, such as a floppy disk; a random access
memory (RAM) 83 for high speed; a hard copy output device 85, such as a
printer; and a touch screen 82 which, in the preferred, embodiment,
operates as the only input device available directly to the user. The
system operates a switching apparatus 89, a switch being the basic on/off
device which the operator uses to control all the valves, the vortexers,
the autodelivery system 90, and the heater 37. Typically, there is a
one-to-one correspondence between devices and switches in the system so
that each device corresponds to a particular switch number. For example,
switches 0-63 may refer to valve numbers 0-63; the heater may be
controlled by switch 64, etc. Also to implement other elements of
automatic control, the microcomputer 81 receives input signals from the
liquid sensors in order to identify the times of gas-liquid and liquid-gas
transitions, and it receives information from a bar code reader 108
located on the autodelivery system, for cross-checking the identification
of amino acids entering the synthesizer.
Shown in FIG. 3 is a top view of the autodelivery system. The system has a
guideway 101 which serves as a track to hold and control the direction of
motion of an array of cartridges, such as cartridge 105. Each of the
cartridges in the array contains an individual protected amino acid, and
is placed in the array in the sequence that is desired in the peptide to
be synthesized. For convenience, the guideway is open for visual
inspection of the array and is oriented to correspond to the peptide with
the carboxyl terminus on the right, which mimics the typical way peptides
are depicted in the literature and facilitates the comparison of the
sequence in the loaded guideway with that of the desired peptide. To hold
the array of cartridges, a pressure block 103 is held against the last
cartridge of the array by a spring reel 107 which is typically implemented
with a steel restorer tape 110 entrained over a pulley 109. This allows
movement of the pressure block along the guideway while still providing a
substantially constant force against the array of cartridges, thus
accommodating arrays of different lengths which correspond to peptides of
different lengths. Additionally, more than one peptide can be synthesized
from a single array of cartridges. On the end of the guideway opposite
pressure block 103 is an ejector system 115 driven by an air cylinder 113,
for holding cartridges in a sampling position 117 such as that shown for
cartridge number 2, and for ejecting the cartridges once the amino acids
therein are educted. Position 111 for cartridge number 1 illustrates the
eject position of ejector system 115, from which the spent cartridge falls
down a shoot (not shown) and is disposed of. When the ejector returns to
its normal position after ejecting, the constant force spring 107, acting
through pressure block 103, forces the next cartridge into delivery
position.
Also included in the autodelivery system is a bar code reader 108. In the
preferred embodiment, each cartridge is labeled with a bar code unique to
the kind of amino acid it contains. When a cartridge progresses down the
guideway to the location of the bar code reader, the reader reads the bar
code label and sends the information to the computer system. If the
computer has been pre-set for a particular polypeptide, it performs a
consistency check to ensure that the cartridge is in the correct position
in the sequence for that polypeptide. If the computer has not been pre-set
for a particular polypeptide, the system runs open loop and the computer
uses the information from the bar code to call the synthesis protocol to
be used for that particular amino acid in the cartridge and to record the
amino acid used. Also, each cartridge contains a stoichiometrically
correct quantity of amino acid.
In order to educt amino acid from a cartridge, the system is provided with
two syringe needles (shown in FIG. 1), a needle 121 for supplying gas
pressure and for venting and a needle 123 for supplying DCM to the
cartridge to dissolve the amino acid and for educting the dissolved amino
acid and DCM. The two needles are typically mounted vertically and
connected to an air cylinder (not shown) for moving the needles up and out
of the way when a new cartridge is moved into place, and for driving the
needles down through the top of cartridge for the mixing and educting
operations.
FIGS. 5a, 5b, and 5c show the details of the typical cartridge 105 used in
autodelivery system. In the preferred mode, the container is constructed
of blown, high density, polyethylene, and has a body portion 130 of
substantially rectangular cross-section capable of holding about 7 ml. It
also has a neck portion 133 onto which is attached a serum-finished cap
135 having an integral septum 137, which acts to provide a positive seal
of the cap to the neck. As illustrated in FIGS. 4a and 4b, the dimension
D1, (.about.0.5 in.) is typically considerably less than dimension D.sub.2
(--1.120 in.) so that a relatively large number of cartridges (up to 50)
can be used in an array on guideway 101 without the length of the array
becoming unwieldy. Also, to promote proper positioning in the array, the
cartridge has two substantially flat surfaces 134 and 136 on each face.
The bottom of the cartridge is formed in the shape of two planes 140 and
141 intersecting at an angle to form a vee-shaped trough. When the
cartridge is in the sampling position in the autodelivery system, needle
123 decends very close to the line of intersection 143 of the two planes,
which corresponds to the locus of points having the lowest elevation in
the cartridge, i.e. in the bottom of the cartridge. The needle being
located near the lowest point in the cartridge helps to ensure that all of
the material in the cartridge is educted, thereby making possible careful
control of stoichiometry. In order to stabilize the cartridge as it sits
in guideway 101, a flange 145 extends across the bottom of the cartridge
in a direction perpendicular to the line of intersection 143. The
vee-bottomed trough and flange 145 make it possible for the cartridge to
stand unassisted in a stable upright position. The cartridge also includes
an indentation 147 around its circumference to promote precise placement
of a bar code label to ensure the accuracy of bar code reader 108 in
reading the label.
FIG. 6 is a table listing the various dimensions of the bottle.
Illustrated in FIGS. 7a, and 7b are cutaway views of a typical liquid
sensor used in the synthesizer. In the top view of FIG. 7b, the device is
symmetric about the centerline CL, so the top half of FIG. 7b corresponds
to the bottom view of the top half of the device, and the bottom half of
FIG. 7b corresponds to the top view of the bottom half of the device. The
device is made up of a clothespin-like tube-holder housing having a top
portion 222 and a bottom portion 220, typically constructed of
glass-filled nylon or plastic, each of which has a groove 229 with a
double curvature extending across the width thereof to accommodate a
translucent tube. The double curvature is provided to enable the top and
bottom portion to positively engage two different tube diameters, which in
the preferred embodiment are typically one-eighth or one-sixteenth of an
inch in outside diameter and constructed of TEFLON.TM.. The top and bottom
portions 222 and 220 are mounted by pegs 212, 213, 214, and 215, to two
substrates, 209 and 211, respectively, which are typically constructed of
printed circuit board material (phenolic). At the end opposite the
tube-holder, the substrates are held a fixed distance apart by two rivets
240 and 241 of substantially the same length. By providing a thickness of
the tube-holder housing, from top and bottom, which is thicker than the
length of the rivets, the substrates provide a spring-like force to keep
the top and bottom portion of the housing together, thereby firmly holding
the tube in groove 229. Also to ensure accurate alignment of the top and
bottom portions, a key arrangement is provided with keys 225 and 226,
located on each side of the top portion which fit into holes 227 (not
shown because of the cutaway in FIG. 7b) and 228 located in bottom portion
220. In the side view of FIG. 7a, bottom portion 220 has been cut away to
reveal a hole 250 in which is located a photodiode 270. Immediately
opposite hole 250 across groove 229 is an identical hole 251 located in
top portion 222 for accommodating a photodetector 271, which is used to
detect the change in intensity of light received from the photodiode when
the interface between a liquid and gas, or between a gas and liquid moves
down the tube held in groove 229, the change in intensity being due to the
difference in focusing of the light rays due to the difference in
refractive properties of liquid and gas. Also, a void 252 is provided in
top portion 222 to accommodate a holder for the photodetector, and a
similar void 253 is provided in lower portion 220 to accommodate a holder
for the photodiode. Similarly a conduit 260 through bottom portion 220 and
conduit 261 through top portion 222 provide paths for the electrical leads
from the diode and detector, respectively, to solder pads 230 and 231
which are located at the ends of electrical runs 233 and 234, and to the
outside generally for the detector signal lead. Power is provided to the
photodiode and the detector via input terminals 235 and 236. Terminals 237
and 238 provide a common ground for both the photodiode and the detector.
Synthesizer Operation
Synthesis of a peptide is initiated by first loading the reaction vessel
with resin, typically to which is attached the first amino acid in the
sequence, and entering the desired amino acid sequence into the computer.
The operator then loads the amino acid cartridges into the autodelivery
system in the linear sequence or chain that corresponds to the amino acid
sequence of the desired peptide.
A cycle of activation begins when needles 121 and 123 puncture the septum
of the first cartridge, and needle 123 injects a calibrated amount of DCM.
Gas sparges from needle 121 are used to mix and dissolve the protected
amino acid in the DCM. After dissolution the protected amino acid is
educted and transferred to the activator vessel. To assure total transfer
of the protected amino acid, a second (and perhaps third) volume of DCM is
added to the cartridge and then transferred to the activator vessel. Then,
based on the amino acid, 0.5 equivalent of DCC in DCM is delivered to the
activator vessel and the solution is mixed by periodic gas burps, e.g.
argon or nitrogen. After a predetermined time interval sufficient for
complete conversion of the amino acid to its symmetric anhydride, the gas
line of valve block 25 is opened and the DCM solution of the PSA is
pressured out through valve block 23 to the concentrator vessel. Frit 17
at the bottom of the activator vessel retains all of the DCU precipitate
that is formed as a by-product in the activation reaction. With software
control, the PSA reaction times can be individually adjusted for each
amino acid to optimize PSA formation and for maximum precipitation of DCU.
After transfer of the PSA/DCM solution to the concentrator vessel, a
volume of DMF is added. The vent on the valve block 33 is then opened and
an inert gas sparge through valve block 41 is commenced to volatilize the
DCM. This is done without significantly reducing the original volume of
DMF, which has a significantly higher boiling point than DCM. Heat is
supplied by band heater 37 to replace heat lost by evaporation of the DCM.
During this solvent replacement process, different maximum internal
temperatures can be automatically adjusted to the unique thermal
stabilities of the various protected amino acid PSA's by the use of
thermistors. Concurrently with the solvent replacement process in the
concentrator, the DCU precipitate in the activator is removed by
successive washings with an alcohol/DCM mixture via the top valve (valve
block 23) and overhead wash nozzle 19. The activator is finally washed
with dichloromethane in preparation for the next PSA reaction. In the
concentrator vessel after the dichloromethane has been removed, the
PSA/DMF solution is pressure transferred from the concentrator vessel to
the reaction vessel which contains the resin-bound alpha-amino-deprotected
growing peptide chain.
The PSA/DMF that has been brought into the reaction vessel from the
concentrator vessel reacts with the deprotected alpha-amino function of
the resin bound peptide for a period of time sufficient for reaction
completion (typically greater than 99%), after which spent reagent and
solvent are washed out by successive solvent washes while using vortex
agitation.
To begin a new cycle of synthesis in the reaction vessel it is first
necessary to remove the alpha-amino protecting group of the last amino
acid which was attached to the chain. In the specific case of t-BOC
protected amino acids, a solution of trifluoroacetic acid (TFA) and DCM,
typically 65% TFA, is pressure transferred to the reaction vessel and
vortex agitation is periodically applied for effective mixing. After a
time sufficient for total removal of the t-BOC-alphaamino protecting
groups (typically about 15 minutes), the fluid is pressured out through
valve block 45 to waste. The resin is then washed rapidly with small
volume increments of DCM introduced either through the top or bottom valve
while vortexing. Neutralization is effected by the introduction of
diisopropylethylamine (DIEA) and DMF or DCM, vortexing, followed by
pressure delivery to waste. Neutralization is usually repeated once. The
resin is then washed by successive additions of DCM (or DMF) in small
volume increments through valve block 45 with the top valve (valve block
43) open to waste, while vortexing (vortexing may be continuous or
intermittent). After thorough washing of the aminodeprotected resin, the
next amino acid PSA/DMF mixture is pressure transferred to the reaction
vessel from the concentrator vessel.
To sample the resin during synthesis or on completion of the peptide, first
valve 55 is opened and line 61 is opened through valve block 45 which is
vented to waste, while valve 67 is kept closed. The reaction vessel is
then pressurized from the top while vortexing, forcing resin and the
reaction solution into sample reservoir 57. This drives resin against the
membrane 59. Valve 55 is then closed, valve 67 is opened, and the waste
line of valve block 45 is closed. DCM is passed back through line 61 from
valve block 45 clearing resin from the membrane and depositing the
resin/DCM mixture in a fraction collector 64. The sample reservoir and its
accompanying tubing is then washed by closing valve 67, venting the
reaction vessel, and transferring DCM through line 61 from valve block 45
through valve 55, and into the reaction vessel.
This integrated system allows for simultaneous operations in the reaction
vessel and in the activator and concentrator vessels. For example,
deprotection, neutralization, coupling, and washing operations can occur
in the reaction vessel at the same time that the next amino acid PSA is
being formed in the activator vessel. The concentrator vessel can be
cleaned at the same time activation is occurring the activator vessel, and
the activator vessel can be cleaned while the concentrator vessel is
engaged in solvent replacement. This simultaneity of operations makes
possible large economies in cycle time. Appendix A provides a more
detailed description of this simultaneity by illustrating the time phasing
of operations in each vessel during a complete process for attaching a
single amino acid.
The system also allows the use of various synthesis methodologies. Although
the approach described above has been for peptide synthesis by t-BOC-amino
acid PSA's, alternative methods using protected amino acid PSA's, such as
F-MOC, could also be readily implemented. Similarly, synthesis could be
implemented by using other active carboxyl species such as mixed
anhydrides, active esters, acid chloride, and the like, utilizing the
activator vessel and concentrator vessel to pre-form the activated
carboxyl species just prior to introduction to the reaction vessel, thus
eliminating the need for storage reservoirs of activated amino acid
species which are maintained throughout the time frame of the peptide
synthesis.
As indicated earlier special coupling procedures are necessary for
asparagine, glutamine, and arginine and can be initiated in the activator
and concentrator vessels. In these cases double couplings with
hydroxybenzotriazole (HOBT) and DCC are generally required, where
equimolar HOBT and DCC in DMF or DMF/DCM are preequilibrated and then
combined with an equivalent of protected amino acid for reaction in the
reaction vessel.
To achieve this result, one approach is to first transfer one equivalent
each of HOBT/DMF and DCC/DCM to the concentrator vessel through valve
blocks 23 and 41. Then two equivalents of protected amino acid from an
amino acid cartridge are transferred to the activator vessel in
appropriate solvents (DMF/DCM). Following that, half of the material in
the activator vessel is transferred by time-pressure control to the
concentrator vessel (containing the pre-equilibrated HOBT/DCC/DMF/DCM) for
activation, after which the activated mixture is transferred to the
reaction vessel. Analogous activation is commenced for the second coupling
near the end of the first coupling cycle by recharging the concentrator
vessel with a second equimolar mixture of HOBT/DCC, followed by addition
of the second equivalent of amino acid from the activator vessel.
Computer Software System
At the most basic level, software control of the apparatus is a matter of
turning valves and other switched devices on and off at the proper times
to achieve the desired flows of the various materials from one container
or vessel to another. At the same time, many of the various steps in solid
phase synthesis are quite repetitive and not extraordinary in number. Such
a situation lends itself conveniently to a more sophisticated control
concept aimed at functional control by the operator rather than having the
operator dictate in detail the workings of individual valves to achieve a
desired result. For example, most often the operator would rather command
the system to transfer the contents of the activator vessel to the
concentrator vessel, rather than formulate a more detailed series of
commands such as: (1) check the concentrator vessel to see if it is ready
to receive; (2) open the vent on valve block 33; (3) open valve blocks 23
and 41 at the connection of the transfer line between the vessels; (4)
open gas valve to pressurize the activator vessel; (5) shut off the valves
after a signal from liquid sensor 39 indicates that the fluid has been
transferred. To achieve this kind of user-friendly approach and still
maintain the capability of totally independent control over each element
of the apparatus, the software control system is implemented through the
touchscreen using a series of menus, which serve to provide the operator
with a wide gamut of possibilities, from one extreme of using the system
in a completely automated mode to the other extreme of operating the
system by switching the individual valves.
The control concept involved is that each individual coupling of an amino
acid to the peptide chain corresponds to three complete cycles: one cycle
in the activator vessel, one cycle in the concentrator vessel, and one
cycle in the reaction vessel. Each of these cycles consists of an ordered
set of timed individual steps, each of which corresponds to a function
which can occur in that vessel. As a general definition, a function can be
considered as corresponding to a named set of switches which are turned on
simultaneously (the normal position of each switch being off). In practice
it is advantageous to number the various functions and to separate them by
vessel. A function for example, might be DCM TO ACTIVATOR. Such a function
requires a particular configuration of open valves in order for DCM to be
delivered to the activator. In a cycle of the activator, this function may
appear several times, e.g. after the bulk of symmetric anhydride has been
transferred to the concentrator vessel it may be advantageous to wash the
activator vessel and DCU precipitate several times with DCM to remove any
residual symmetric anhydride. Each time this function occurs it will
correspond to a different step in the reaction cycle occurring in the
activator vessel, and similarly for other functions which are required in
each cycle. The net result is that each cycle in a vessel is a series of
steps, with each step corresponding to a function associated with that
vessel. A typical list of functions is provided in Appendix B. To better
illustrate this concept, the individual control menus will now be
described.
FIG. 8 shows the main menu of the system as it appears on the touchscreen
which corresponds to the bottom of a tree (shown in FIGS. 17aand 17b) of
various other more detailed menus. Each outlined block corresponds to an
area on the touchscreen by which the series of menus in that tree is
accessed. For example, touching the screen at the block labeled "PEPTIDE
SEQ. EDITOR", initiates another display, FIG. 9, listing all of the amino
acids, and allows the selection and display of the order of amino acids
from N to C terminus appearing in the peptide to be synthesized by simply
touching the block containing the name of the desired amino acid in the
desired sequence.
Touching the screen at the block labeled "PEPTIDE CHEMISTRY EDITOR", when
the main menu is displayed brings up the PEPTIDE CHEMISTRY EDITOR menu,
FIG. 10, which allows the operator to create or edit either cycle
specifications in a particular vessel or the run file specification. As an
example, if it is elected to create or edit a new activator cycle, a CYCLE
EDITOR menu corresponding to the activator vessel appears on the screen.
(See FIG. 11.) This menu enables the operator to change the order of the
functions involved in each cycle of the activator, and similarly for menus
corresponding to the other vessels.
Generally, a given cycle has three time fields associated with it: a
required primary time, "TIME"; a time based on detector measurements, "MIN
TIME" and "ERR MODE"; and an additional time added for particular sets of
steps in the reaction vessel, "ADD TIME". TIME has several purposes. When
liquid detection is not specified for the step, TIME is the total time for
the step. When liquid detection is specified, the primary time is the
"time out" or maximum time allowed before continuing regardless of whether
or not the appropriate transition was detected by the liquid sensor. Also,
when liquid detection is specified, the user must specify a minimum time,
MIN TIME, before the appropriate transition (liquid to gas or gas to
liquid) is to registered. It is important to note another effect of TIME
when detection is specified. When the sensor indicates the correct
transition has occurred after the specified minimum time, the indicated
function will be terminated, i.e., the switches for that function will be
turned off. However, the next step will not be initiated until the primary
time has elapsed. This is necessary to ensure proper alignment of the
cycles in each vessel to obtain optimum throughput. ERR MODE is used in
the event that detection is specified and the specified transition is not
seen before the primary time has elapsed, e.g. if a valve is blocked or a
particular reservoir is empty. This mode can be used to trigger an alarm
or, in some cases, to effect a non-disastrous termination of the
synthesis. ADD TIME refers specifically to the reaction vessel only, and
corresponds to the amount of time (in tenths of a second) to be added to
each of the three previous fields as a function of amino acid number in
the sequence of the peptide being synthesized. Since the occupied volume
in the reaction vessel increases with each additional amino acid coupling,
the step times in the reaction vessel also increase. For example, if the
value 10 is entered into this field, one second (10/10ths) will be added
to the primary time for the second amino acid to be coupled in the peptide
sequence. For the third amino acid, the time would be increased by two
seconds, and so forth.
If it is desired to edit a RUN FILE, EDIT OLD in the PEPTIDE CHEMISTRY
EDITOR is selected, displaying the RUN FILE EDITOR menu shown in FIG. 12.
The table displayed therein corresponds to what is called the static run
file. This file designates three cycles (one each for the activator
vessel, the concentrator vessel, and the reaction vessel), for each of
twenty six amino acid possibilities, and allows independent adjustment of
the concentrator temperature for each amino acid. The twenty six amino
acid possibilities provided are comprised of the twenty standard amino
acids, an additional four for specialized use as might be desired by the
operator, and a BEGIN cycle and an END cycle to allow independent control
of these points in the synthesis. This static run file is the result of
specifying the chemistry for each cycle through the various CYCLE EDITORS
which have already been described). In addition, all cycles designated by
a particular run file must be resident on a disc currently installed in
the system, since the RUN FILE EDITOR will only allow choices from a list
of resident cycles. Also, each of the three reaction vessels may
synthesize from a different (or the same) run file, since, at the time a
run is set up, the run file is specified for the particular vessel in the
REACTION VESSEL MONITOR (which will be discussed later). Typically, the
operator may create, edit, and store a number of run files on a single
disc (up to 20). It should also be noted that although the system is
designed to permit a different cycle for each amino acid possibility, it
has been found in practice that not nearly that many cycles are needed to
provide efficient operations.
The next menu which will be discussed is the VESSEL FUNCTION EDITOR. This
menu is accessed from the main menu, and operates at the most basic level
of the synthesizer. It involves the individual instructions required to
accomplish a particular function in a particular vessel. For example, if
it is desired to change the definition of a function or add or delete
functions which are to occur in the activator vessel, the FUNCTION EDITOR
shown in FIG. 13 corresponding to the activator vessel is called. Here,
the entire set of functions for the activator can be reviewed and changed.
As indicated earlier, a function is a named set of switches turned on
simultaneously. These functions usually create a chemical flow path
through the system. For example, the function "DCC TO ACTIVATOR" may be
defined as switches 114 (DCC delivery valve), 119 (pressurize DCC bottle),
and 125 (open activator to waste). Also some functions describe mechanical
or electrical actions only, such as "heater on", (one switch). The system
is organized into three types of functions: ON/OFF, TOGGLE ON, and TOGGLE
OFF, specified as type 0, 1, and 2, respectively. An ON/OFF function turns
switches on for a given step in a cycle only. At the end of that given
step, all switches for that function are cleared (turned off) before the
next function is activated. A TOGGLE ON function directs the machine to
turn on certain switches and leave them on until a corresponding TOGGLE
OFF function turns them off. Subsequent functions will not affect the
state of the switches turned on by the TOGGLE ON function. Hence, during
creation and editing of functions, it is important that all TOGGLE ON
functions have a corresponding TOGGLE OFF function. Examples of TOGGLE ON
functions are "Heater on" for the concentrator vessel, or "VORTEX ON" for
the reaction vessel.
Calling the REACTION VESSEL MONITOR from the main menu presents the screen
shown in FIG. 14, which lets the operator set up and monitor the
synthesis. Two response fields permit the operator to select between two
modes of operation, a first mode which automatically controls the
apparatus based on the input to the PEPTIDE SEQ. EDITOR and a set of
preprogrammed cycles called the dynamic run file; and a second mode which
operates based only on which cartridges (kinds of amino acids) are loaded
into the autodelivery system and a set of preprogrammed cycles from a
designated static run file. If the first mode is chosen, the computer
initiates a question as to whether the operator wishes to change the
dynamic run file. If so, the operator responds in the affirmative and a
screen as shown in FIG. 15 is displayed, corresponding to an editor for
the dynamic run file. This file is generated initially internally when the
operator enters the desired peptide sequence into the PEPTIDE SEQ. EDITOR.
It is simply a table listing the sequence of amino acids in the order to
be synthesized, with the designated cycles for each amino acid as has
already been programmed from the designated static run file. This editor
is designed so that the operator can alter the cycles used for particular
amino acids in the sequence depending on where they are located in the
peptide chain, so that the operator has position dependent control over
the chemistry. Unlike the static run file, the dynamic run file is not
stored on the disc.
Following mode selection in the REACTION VESSEL Monitor, a series of
questions is then generated to ensure that the apparatus is properly set
up to begin operations, e.g. checks are made as to whether the reaction
vessel is loaded, and whether the autoloader has the desired number and
order of amino acids.
Following that series of inquiries, the final inquiry is whether to begin
or to stop operations. Also as part of the function of the REACTION VESSEL
MONITOR, the status of the reaction vessel is displayed, including the
name of the activated amino acid that is currently undergoing reaction,
the time that the synthesis of peptide was initiated, and the time when
the synthesis was completed. In addition, the current coupling is
displayed and the sequence of couplings already completed can be displayed
and reviewed by scrolling back and forth on the screen.
Calling the CYCLE MONITOR from the main menu brings up the screen shown in
FIG. 16. Here the current status of each vessel is displayed in real time
in terms of several parameters. On the first line is listed for each
vessel the particular number of the amino acid in the sequence of the
peptide which is currently active in that vessel, along with the
abbreviation of the name of the particular amino acid. The second line
lists the particular cycle name currently ongoing in each vessel. The
third line lists which step in the sequence of steps of the particular
cycle is currently being carried on in each vessel. The fourth line lists
the number of the function and the function name corresponding to the
particular step listed for that vessel. Also the elapsed time into each
cycle is listed as is the status of each of the liquid sensors.
Several other menus may also be selected from the main menu under the
general category of MACHINE/STATUS SELECTIONS. These include: RESERVOIR
STATUS which relates to the fact that the apparatus monitors the volume
used from each reservoir during each cycle, so that when a reservoir runs
low an alarm is registered to inform the operator of the problem;
INSTRUMENT CONFIG. which is used to display and set the configuration of
the machine itself, e.g. time of day, power line -120V at 60 Hz, etc.;
SYSTEM SELF TEST which tests all of the electronic functions to the extent
practicable; CONTROL AND TEST which allows manual control of the apparatus
to the extent that the operator can manipulate each valve and switch, one
at a time, in order to facilitate debugging and to enable manual
intervention of synthesis if necessary; and DISK UTILITIES which allows
the operator to carry out customary disc functions necessary to the
operation of a computer system, e.g. setting up files, purging files, and
renaming files.
Another characteristic of the apparatus which is controlled by the software
is the simultaneity of operations in the reaction vessel, the concentrator
vessel, and the activator vessel. By noting the various times required to
carry out each step of a cycle in each vessel, an automated optimization
scheme, hereinafter called a cycle compiler, has been developed to provide
maximum efficiency in the synthesis of each particular peptide given a
particular chemistry. To achieve this efficiency it is important to
recognize that for each vessel certain events (functions) in time, in each
cycle, determine when transfers can take place and when the vessel is
ready to begin the process for another transfer. For the activator vessel,
these functions correspond to the beginning of a transfer, BOTA; the end
of a transfer, EOTA. particular peptide, given a particular chemistry. To
achieve this efficiency it is important to recognize that for each vessel
certain events (functions) in time, in each cycle, determine when
transfers can take place and when the vessel is ready to begin the process
for another transfer. For the activator vessel, these functions correspond
to the beginning of a transfer, BOTA; the end of a transfer, EOTA.
Similarly for the concentrator cycle, the key functions include identifying
when the vessel is ready to receive, RC; as well as the beginning of a
transfer, BOTC; the end of a transfer, EOTC. For the reaction vessel,
these functions delineate when the vessel is ready to receive, RR. Using
these concepts, a graphic representation of a cycle as it takes place in
each vessel can be depicted as illustrated in FIG. 18. From this figure,
it is apparent that one of the first criteria for operation is that
T.sub.BOTA =T.sub.RC and T.sub.BOTC =T.sub.RR ;
i.e. the concentrator vessel must be ready to receive when a transfer from
the activator vessel is begun, and the reaction vessel must be ready to
receive when a transfer from the concentrator vessel is begun. The next
step then is to determine in which vessel operations should begin first,
in order to optimize the process. This can be accomplished by calculating
the left side delays (from FIG. 18 ) for each vessel: T.sub.DLA,
T.sub.DLC, and T.sub.DLR. These left side delays pertain to the waiting
time allowed before preparing the particular vessel for the function that
is to take place there, e.g. for the activator vessel, some waiting time
may be allowed before transferring amino acid into the vessel and creating
the symmetric anhydride, and for the reaction vessel, there may be some
waiting time allowed before beginning the deprotection of the resinbound
peptide chain. Once these left side delays are calculated, the shortest
delay (i.e. the longest preparation time) then corresponds to the cycle
which should begin first, and the master time should be equal to zero for
that cycle. These various delays can be calculated according to the
following equations. First define
T.sub.MA .ident.(T.sub.EOTA -T.sub.BOTA)+(T.sub.BOTC -T.sub.RC), and
T.sub.ML .ident.MAX (T.sub.BOTA, T.sub.RC, T.sub.RX),
where T.sub.RX .tbd.T.sub.RR -T.sub.MA. (See FIG. 18 for a physical
interpretation of T.sub.MA and T.sub.ML)
Then T.sub.DLA =T.sub.ML -T.sub.BOTA,
T.sub.DLC =T.sub.ML -T.sub.RC, and
T.sub.DLR =T.sub.ML -T.sub.RX.
For example, setting the master time equal to zero at the beginning of the
cycle shown in FIG. 18 might yield delay times such as T.sub.DLA =0,
T.sub.DLC =5, and T.sub.DLR =3, i.e. the activator vessel would begin
first at time T=0 the reaction vessel then would begin 3 seconds later,
and the concentrator vessel would begin at T=5 seconds.
The next requirement for optimal throughput involves calculating the
minimum separation of these three cycles with the three cycles of the next
coupling sequence To calculate this minimum, it is important to evaluate
the right side delays for the first cycle:
T.sub.DRA =T.sub.BOX -T.sub.DLA -T.sub.AT ;
T.sub.DRC =T.sub.BOX -T.sub.DLC -[T.sub.CT +(T.sub.EOTA -T.sub.BOTA)];
and T.sub.DRR =T.sub.BOX -T.sub.DLR -[T.sub.RT +(T.sub.EOTC -T.sub.BOTC)];
##EQU1##
and where T.sub.AT, T.sub.CA, and T.sub.RT correspond to the total time
for the activator cycle, concentrator cycle, and reaction vessel cycle,
respectively. These right side delays must then be combined with the
various left side delays for the next cycle to arrive at a minimum
separation for the three cycles, i.e.
T.sub.S.sbsb.c2-cl =MIN[(T.sub.DRA +T.sub.DLA2), (T.sub.DRC +T.sub.DLC2),
(T.sub.DRR +TD.sub.LR2)],
where T.sub.S.sbsb.c2-cl is the minimum separation between the first cycle
and the second cycle, and T.sub.DLA2, T.sub.DLC2, and T.sub.DLR2 are the
left side delays of the second cycle (calculated using the same technique
as for the left side delays in the first cycle). This minimum separation
can then be translated into the waiting times required to start the second
cycle in each of the vessels, i.e.
T.sub.WA2 =T.sub.DLA2 +T.sub.DRA -T.sub.S.sbsb.c2-cl
T.sub.WC2 =T.sub.DLC2 +T.sub.DRC -T.sub.S.sbsb.c2-cl, and
T.sub.WR2 =T.sub.DLR2 +T.sub.DRR -T.sub.S.sbsb.c2-cl
where T.sub.WA2, T.sub.WC2, and T.sub.WR2 are the waiting times for the
activator vessel, the concentrator vessel, and the reaction vessel,
respectively, from the last step of the first cycle to the first step of
the second cycle. In order to achieve optimum efficiency one of T.sub.WA2,
T.sub.WC2, and T.sub.WR2 must be zero, as was the case for left side
delays in the first cycle. FIG. 19 shows the results of the above
calculation for a series of three cycles.
Utility of the Invention
The ability to deliver amino acid symmetric anhydrides of predetermined
integrity to the reaction vessel enables most coupling reactions to
proceed in yields exceeding 99% using just single couplings. Because of
this very high yield at each step, it is possible to synthesize peptides
with very high overall efficiency fully automatically. The combination of
activator vessel and thermally jacketed concentrator vessel make it
possible to study and develop optimal conditions for maximal symmetric
anhydride formation for individual protected amino acids, by individually
modifying stoichiometry, reaction times in DCM, temperature during DMF
replacement of DCM, and the time frame during which the solvent
replacement process is executed. Thus, for each type of amino acid it is
possible to automatically implement conditions which will reproducibly
deliver a maximal quantity of symmetric anhydride to the reaction vessel
containing the peptide resin.
In the prior art with peptide synthesizers having no ability to form
preactivated amino acids, the user is obliged to stop at each reaction
cycle after coupling to monitor the extent of coupling. Then with the
objective of improving the yield, second, or even third couplings can be
effected in cases where the first coupling reaction was relatively
inefficient. The result is automation of a single cycle of synthesis at
the preclusion of automation from cycle to cycle. Alternatively, existing
synthesizers can be used automatically from cycle to cycle by utilizing
multiple couplings with in situ activation in the reaction vessel
containing the peptide resin. This results in relatively inefficient
couplings since the activation method cannot be uniquely optimized for
individual amino acids in the presence of the reacting amino group of the
growing peptide chain.
The results shown in FIGS. 20a and 20b illustrate the relative advantage of
the claimed apparatus for the synthesis of the decapeptide Acyl Carrier
Protein (65-74). The uppermost line is a graphical representation of the
individual cycle yields for each amino acid addition during the peptide
chain assembly as performed on the apparatus of the invention. Except for
glutamine (symbolized by Gln), all amino acid additions were accomplished
by a fully automated sequence of single couplings: glutamine requires a
special chemical protocol well known in the art that consists of double
coupling with HOBT (hydroxybenzotriazole) activation. All other couplings
were done with Boc-amino acid symmetric anhydrides.
The lower line depicts results of the synthesis of the same peptide on a
Beckman 990 Peptide Synthesizer using Boc-amino acid symmetric anhydrides
pre-formed manually, off the instrument. Reference: Reza Arshady, Eric
Atherton, Derek Clive, and Robert C. Sheppard J. Chem. Soc. Perkin Trans
1, (1981) 529-537. The results demonstrate that fully automated synthesis
was precluded by the need to manually preform the symmetric anhydrides,
and that the manually preformed symmetric anhydrides were not optimally
formed.
Some peptide amino acid sequences demonstrate unique, sequence specific
coupling problems where even second and third couplings, typically in DCM,
fail to effect greater than 99% coupling yields, irrespective of the
activated form of the amino acid (see W. S. Hancock, D. J. Prescott, P. R.
Yagelos, and G. R. Marshall, J. Org. 38 (1973) 774). However, those
skilled in the art recognize that sequence dependent incomplete couplings
performed in poor solvents such as DCM would be much improved if performed
exclusively in more polar solvents such as DMF (see S. Meister, S. B. H.
Kent, Peptides, Structure & Function, Proceedings of the 8th American
Peptide Symposium, pps. (103-106). The Acyl Carrier Protein (65-74)
decapeptide is such a known problem sequence and the excellent results
achieved on the claimed apparatus by coupling with Bocamino acid symmetric
anhydrides in DMF demonstrate one of the chief advantages of the
apparatus: optimal formation of the activated amino acid in DCM, but
coupling of the activated amino acid in DMF. This automatically executed
method affords a general solution to the synthesis of problem peptide
sequences. PG,41
APPENDIX A
__________________________________________________________________________
Example of a typical Amino Acid Pre-formed
Symmetric Anhydride Cycle
For all amino acids except GLN, ASN and ARG.
Each line described is a separate type of function.
A function opens specific valves for a given operation.
Rx Vessel Concentrator Activator
__________________________________________________________________________
If this is the first cycle of a Advance Fraction collector (for
series then the resin is washed Resin collector)
3 times with DCM (all washes include Needle down
vortexing)* DCM to cartridge
Mix cartridge
Wait to dissolve amino acid
##STR6##
##STR7##
Measure DCC
DCC to Activator
Add DCM to RV (.about.1.5 ml) Reaction time in Activator of
about
Add TFA to RV (.about.3 ml) 10 min. (depending on amino acid)**
-
(This makes about a 65% series of waits & mixing during
this
dilution of TFA in RV) time - if mixing does not occur at
Drain RV about 30 second intervals, then
the
Repeat above additions. precipitate forms a plug that will
65% TFA is then left in RV for not break up). This reaction is
.about.15 min with mixing - for faster in DCM than DMF.
deprotection
##STR8##
Rinse valve block 4 times
Transfer contents of Activator
Rinse activator & precipitate
to eliminate TFA - blow back
to concentrator with DCM from top of Activator
twice
gas into TFA bottle so that & transfer each rinse to
concentrator.
TFA is not sitting in line
Blow nitrogen gas through contents.
A combination of DCM and methanol
are
against valve block -
This cools the mixture rapidly.
then added to yield approximately
a
Vent TFA bottle so its not
Heat the concentrator vessel
6/4 ratio to dissolve the
under pressure. so that the temperature doesn't
Dicyclohexylurea (precipitate).
During the 15 min. TFA time the resin
exceed a certain point (depending
This dissolving takes a few
minutes
sample line is rinsed with DCM twice.
on amino acid). with mixing in between.
At the end of 15 min. the RV is
At various time intervals add DMF
Drain activator to waste.
drained. to the concentrator (.about.1 ml at a
Rinse activator from top with DCM
##STR9## time, so that the DCM is effectively being replaced
with DMF. This process will take from 5-15
to eliminate the methanol. This is
done in a series of rinses & drains
ith mixing in between.
##STR10##
##STR11## The activator then waits for the
next cycle
Mix with top vent open so that top line is washed (TFA is neutralized)
Add DIEA & DMF to the resin sample line (to neutralize any TFA that may
have entered the line). Drain RV Repeat DIEA, DMF Drain
##STR12##
##STR13##
##STR14##
##STR15##
##STR16## Rinse concentrator with DMF from top (twice) &
transfer each wash to RV
Reaction time of 30 min.
Wash concentrator thoroughly with
DCM - series of washes from top
and drain.
(Rinse resin sampler line during
this time periodically.)
Wait for next cycle.
At end of Rx time, drain RV
##STR17##
Before last drain of RV -
remove resin sample from RV and
transfer to fraction collector
__________________________________________________________________________
DCM = Dichloromethane; DMF = N,NDimethylformamide;
DCC = N,NDicyclohexylcarbodiimide; TFA = Trifluoroacetic acid;
DIEA = Diisopropylethylamine
NOTES:
Washes of RV are 10 ml each. Start vortexing at beginning of addition of
wash solvent. This helps to break up any resin agglomeration. Option:
washing from top or bottom of RV.
Valve blocks are washed with DCM and blown out with nitrogen after
transfers and deliveries of certain reagents.
*The resin used is typically loaded PAMResin (1% styrene Divinyl benzene
resin). The carboxy terminal amino acid is loaded onto the resin through
an organic linker. Typically, 0.5 mmoles of loaded resin (.about.1 mmole
amino acid/gram polystyrene) is used.
**10 min. is used for all aminoacids except GLN, ASN, and ARG. However,
individual variations can be incorporated at the request of the operator.
***For Tyrosine and Lysine approximately 7 min. is typical; and for all
others, except GLN, ASN and ARG, 15 min. is typical.
______________________________________
APPENDIX B
LIST OF POSSIBLE FUNCTIONS FOR PEPTIDE
SYNTHESIZER
Key: T = top valve block; B = bottom valve block;
0 = off; 1 = on
______________________________________
Reaction Vessel Functions
DCM to waste (T)
Extra to waste (T)
gas VB (T)
Vent RV (T)
Pressurize RV (T)
DCM to RV (T)
DCM to waste (B)
DMF to waste (B)
DIEA to waste (B)
DIEA Pressure
TFA to waste (B)
gas VB (B)
Drain RV (B)
Blow back into TFA bottle
Vent TFA bottle
TFA Pressure
Manifold Pressure
Neutralizer Pressure
Neutralizer Delivery
Vent RV (B)
Pressurize RV (B)
DCM to RV (B)
DMF to RV (B)
DIEA to RV (B)
TFA to RV (B)
Gas to RV(B)
Resin Sampler (RV functions)
take sample
DCM to RV
DCM to fraction collector
DMF to RV
DMF to fraction collector
Gas to RV
Gas to fraction collector
DIEA to RV
Misc RV Functions
Vortex (0,1)
Fraction collector advance
Concentrator
DMF to waste (T)
DCM to waste (T)
Gas VB (T)
Pressurize Conc (T)
Vent (T)
Drain
DCM to Conc (T)
DMF to Conc (T)
DMF to waste (B)
Extra to waste (B)
Gas VB (B)
Pressurize Conc (B)
Vent (B)
DMF to Conc (B)
Extra to Conc (B)
Manifold Pressure
Transfers
Conc to RV
DMF transfer line
Extra to transfer line
Gas transfer line
Misc Conc Function
Heater (0,1)
Activator
MeOH to waste (T)
DCM to waste (T)
Gas VB (T)
Pressurize Act (T)
Drain Act
Vent Act (T)
DCM to Act (T)
MeOH to Act (T)
DCM to waste (B)
Extra-1 to waste (B)
DMF to waste (B)
Extra-2 to waste (B)
AA to waste (B)
Gas VB (B)
Measure DCC
Measure HOBT
Manifold Pressure
Pressurize Act (B)
Gas to mix (B)
DCM to Act (B)
DMF to Act
DCC to Act
HOBT to Act
Amino Acid to Act
Extra-1 to Act
Extra-2 to Act
Cartridge Delivery System
DCM to cartridge
DMF to cartridge
Extra-1 to cartridge
Extra-2 to cartridge
Pressurize cartridge (sample needle)
Pressurize cartridge (vent needle)
Mix cartridge
Vent cartridge (sample needle)
Vent cartridge (vent needle)
Transfers
Act to Conc
DCM transfer line
DMF transfer line
Extra-1 transfer line
Extra-2 transfer line
Gas transfer line
Misc Act Functions
Needle up & down (0,1)
Cartridge Eject
______________________________________
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